Chitin, the structurally simplest glycosaminoglycans, which is a β-1,4-linked linear homopolymer of N-acetylglucosamine, distributes widely in arthropods and several microbes [1]. In insects, as an insoluble polysaccharide, chitin is a momentous structural component of exoskeleton, trachea and the peritrophic matrix (PM) as well as salivary gland, eggshells, and muscle attachment points [2]. Due to the rigidity and chemical steadiness of chitin, insects build a rigid exoskeleton to protect themselves from environment injuries and pliable PM to keep their midgut safe from mechanical damage and pathogen infections. However, chitin is also the key factor that constrains the growth and development of insects and in order to grow, insects have to periodically shed off their old exoskeleton and turn over PM, and then the new version are rebuilt for defending themselves [3,4].

Chitinase (EC 3.2.1.14, endochitinase) is a kind of glycosyl hydrolases that hydrolyze the β-1,4-glycosidic linkages in chitin. In addition, they extensively exist in nature as they are found in species across all kingdoms, while they function very differently and are mainly involved in digestion, arthropod molting, defense/immunity, and pathogenicity [5]. Insect chitinase is divided into Glycoside hydrolase family 18 (GH18; PFAM database accession: PF00704, http://pfam.xfam.org/ accessed date, 14 March 2021) chitinase-like superfamily which include chitinases with catalytic activity as well as those lacking chitinase activity, such as imaginal disk growth factors (IDGFs), endo-β-N-acetylglucosaminidases (ENGases), stabilin-1 interacting chitinase-like proteins (SI-CLPs) and chitolectins [6]. The proton donor glutamic acid in the active site of chitinase is important for the hydrolysis of β-1,4-glycosidic bond in chitin and its substitution in the enzymatically inactive GH18 members accounts for the lack of chitinolytic activity, even if they may still do well in binding chitin with high infinity [6,7]. Chitinase is critical in helping insects shed off old cuticle and PM turnover. During molting, insects use chitinases to hydrolyze the structural polysaccharide in their exoskeletons and gut linings and digested the insoluble polymeric chitin into soluble, yielding low molecular mass multimers of N-acetyl-beta-D-glucosamine (GlcNAc), such as chitotetraose, chitotriose, and chitobiose [1,8,9,10].

Numerous chitinases and chitinase-like proteins in different orders of insects have been annotated and characterized with the help of functional genomics, which indicates that there are rather large and diverse groups of genes encoding chitinase-like proteins. Based on the difference in primary structures and domain architectures, insect chitinases have been classified into eight phylogenetic groups (group I–VIII) which are commonly adopted [2,5] and two additionally identified groups, group IX and X, makes it into ten groups [11]. The systematic analysis of biological functions of chitinases and chitinase-like proteins in Tribolium castaneum suggested that chitinase genes in group I and II are engaged in insect exuviate and egg hatching, chitinase genes in group III mainly involved in regulating abdominal contraction and wing expansion, while the imaginal disk growth factor genes in group V have little relation to cell proliferation or differentiation of the imaginal disk but needful for adult emergence [12]. Chitinases and chitinase-like proteins in Nilaparvata lugens, one of the typical hemimetabolous insects, were also studied for function analysis by RNA interference experiments. Five genes were found to be involved in N. lugens moulting and resulted in lethal phenotypes after RNAi-treated. Similar to T. castaneum, five genes related to moulting in N. lugens are either from group I, group II, or group III, except one gene which was tentatively placed in group IV [13]. In Acyrthosiphon pisum, there were also several chitinases which could not be clustered [4]. More chitinase sequences need to be analyzed, as there are still chitinases in these hemimetabolous insects that could not be clustered into a certain group. From this point of view, the inadequate annotation data on chitinase-like genes of different insect species may account for the inability to classify certain genes. Hence the genome-wide identification of chitinase-like gene family in more insect species is necessary for accelerating the classification of insect chitinases.

The intake of double-stranded RNA (dsRNA) by direct injection or oral feeding leads to RNA interference (RNAi) in insects, which provides an efficient tool for gene function analysis [13,14,15,16]. In this study, we applied a nanomaterial-promoted RNAi method which previously reported to improve dsRNA penetration through body wall and RNAi-induced mortality in Aphis glycines [17]. It is always necessary to have concern for the safety of non-targets when RNAi-based method of controlling pests is to be applied in the field. As we know, dsRNA can specifically target gene transcript without affecting non-target species, which on the one hand ensures the safety of non-targets. On the other hand, research showed that silencing of the aphid hemocytin gene (Hem) by a nanocarrier-promoted RNAi method dramatically decreased aphid population density, while the cell survival ratios of S2 cells with the same treatment was more than 96%, which demonstrates the safety of the RNAi method to non-targets [17]. Such transdermal delivery system is convenient to operate and of high efficacy as well as safe to non-targets, so it may provide a promising tool to perform RNAi in the laboratory or field [17,18,19,20].

As one of the hemimetabolous insects, Bemisia tabaci (Gennadius) (Hemiptera: Aleyrodidae) is a notorious pest which causes massive damage to crops in temperate climate around the world [21]. It uses specialized stylet to digest nutrients from plant phloem and transmits numerous plant virus causing severe diseases [22,23]. Research indicates that B. tabaci is a species complex comprising at least 24 cryptic species, among which Mediterranean (MED) and Middle East-Asia Minor 1 (MEAM1) are the most invasive and destructive [21,24,25]. MED and MEAM1 are morphologically indistinguishable and differ in host preference, virus transmission, and insecticide resistance [26,27,28]. Besides, MED is recognized as more resistant and able to develop stronger resistance to insecticides than MEAM1 [28,29], which is assumed to be a main reason for the displacement of MEAM1 by MED in many areas. Particularly in China, MED has displaced MEAM1 since 2005, apparently due to the frequent use of insecticides over past decades [30,31]. More severely, despite chemical control with insecticides remaining effective in controlling B. tabaci, the prevalent use of insecticides has resulted in emerging resistance to many classes of insecticides worldwide, even causing a serious threat to agricultural system, food safety and public health [32,33,34,35,36,37,38,39,40,41]. To maintain sustainable pest management, therefore, new ideas for B. tabaci control need to be developed.

Chitinases play an important role in regulating insect growth and development, which might be a promising target for insecticide-based control of B. tabaci [8]. Although several genes in MEAM1 genome have been annotated as chitinases [42]; however, genome-wide characterization of chitinase-like genes in MED B. tabaci remains unknown to date. In this study, we searched the MED B. tabaci genome [43] to identify genes encoding chitinases and chitinase-like proteins and annotated their cDNA sequences. The gene architectures, phylogenetic relationships, expression patterns and primary biology function analysis of chitinases were analyzed to elucidate chitin metabolism in whitefly growth and development.

2. Current Insights

In nature, chitinase is comprehensively distributed and serves as a generalist which engages in digestion, arthropod molting, defense/immunity, and pathogenicity [5]. Besides, as for insects, chitinases are essential for growth and development across their lifetime. Therefore, understanding of insect chitinase biology is of quite importance for providing promising targets of pest control. In this study, we identified 14 chitinase-like genes in a disastrous agriculture pest B. tabaci for the first time and functionally demonstrated that some chitinases may play a vital role in B. tabaci juvenile exuviation, which armed our arsenal to cope with such intractable pest by interfering the expression of chitinases.

The increasing availability of genomic data from different insect species have greatly accelerated the genome-wide annotation of various gene families, which also helped the identification of chitinase-like genes in B. tabaci. By searching and screening of the B. tabaci genome, 14 genes encoding chitinases and chitinase-like proteins were first identified. It was consistent with the two hemimetabolous sap-feeding insects (N. lugens and A. pisum) that B. tabaci had a relatively small number of chitinase-like proteins when compared to other insect species, especially those in Diptera and Coleoptera [4,10,13]. It may be explained that hemimetabolous insects, such as B. tabaci, undergo an incomplete metamorphosis which is much milder and fewer enzymes and less energy are needed to help reconstruct the new integument [4]. In addition, this highly simplified morphology and auxanology of whitefly nymphs is in tune with their sessile feeding habit [56].

Interestingly, among the three hemimetabolous sap-feeding species, B. tabaci exhibited the most chitinases, which was two more than N. lugens and five more than A. pisum. Molecular phylogenetic analyses revealed that there were three B. tabaci IDGF genes (BtIDGF1-3) which were clustered in Group V, wheras N. lugens and A. pisum had only one IDGF separately. Besides, three genes (BtCht4, BtCht8 and BtCht9) in B. tabaci were failed to be clustered and tentatively placed in group IV; however, for N. lugens and A. pisum, there was only one gene respectively. These discriminations may account for the difference in gene quantity of chitinases between B. tabaci and the other two hemimetabolous insects. It was worth mentioning that NlCht3 and ApCht8 were previously placed in group IV while now could be clustered with BtCht3 and classified as group X genes (Figure 1). BtCht3 has an N-terminal signal peptide ahead of the GH18 catalytic domain followed by two very closely spaced tandem chitin-binding domains and a very long C-terminal stretch ending with a third CBD (Figure 3), which is in consistence with previous studies of genes in group X [11]. However, BtCht4, BtCht8, and BtCht9 along with ApCht7 were still tentatively divided into Group IV because they could not be clustered into other groups. In this case, with increasing numbers of chitinase-like genes annotated in different insect species, it is likely that members in group IV probably would be divided into some other brand-new groups, and the potential functions of these genes should be noticed and studied.

In this study, we explored gene expression patterns of each chitinase and chitinase-like genes in B. tabaci via qRT-PCR. Results revealed that six genes were highly expressed in adult stage and most of them do not have chitinase catalytic activity since the key motif of CR_Ⅱ is mutated (Figure 3 and Figure 5). Among these genes, there are three BtIDGFs and they all shared a similar expression pattern. IDGFs were reported to have multiple functions in insect and mammalian cells, involving in regulation of cell proliferation and acting as chitolectins which interact with cell surface receptors [57,58]. The D. melanogaster IDGF2 was turned out to be a trophic factor involved in energy balance, detoxification, and innate immunity, which promoted cellular and organismal survival of D. melanogaster [59]. IDGF4 in Bactrocera dorsalis played an essential role in regulating its development and temperature adaptation [60]. Although functionality of IDGFs in B. tabaci has yet to be determined, it is implied by this research that IDGFs played essential roles in adult performance. In addition, it might be inferred that BtIDGFs also have some important roles in detoxification of xenobiotics, which associates with the severe resistance of B. tabaci to kinds of insecticides [29,61,62].

Egg and Early nymphal stages of whitefly were considered less detrimental to crops since their limited feeding capacity [29]. In addition, it is sensible and rational to disturb the normal development of whitefly in their early nymphal stage to hold back further loss. Therefore, three chitinase-like genes respectively from Group I, Group II and Group III, which were highly expressed in nymphal stages, were selected as targets for analysis of their potential roles in nymphal development. As results showed, silencing of BtCht10, BtCht5 and BtCht7 led to a significant higher rate of death over ten days of recoding and fewer nymphs survived a molting course in the gene-silenced groups than control (Figure 6). These results were in consistence with previous studies, which clarified that chitinase-like genes from Group I, Group II, and Group III were of key importance in old cuticle degradation [1,12,13]. These results along with previous research, enriches our knowledge about how insect chitinases are involved in chitin synthesis and degradation so that certain enzymatic steps can be perturbed and potentially act as targets of pest control [63]. Consequently, BtCht10, BtCht5, and BtCht7 are as key genes involved in B. tabaci chitin degradation, which provides a promising idea of B. tabaci management by interfering the expression of these representative genes.

3. Conclusions

In summary, 14 chitinase-like genes were identified in B. tabaci genome and these genes were divided into ten distinct groups through phylogenetic analysis, by which there were no B. tabaci chitinase-like genes clustered in group IX. Then, pattern expression analysis showed that these genes displayed rather different models in disparate developmental stages, which may be associated with their discrepant biological functions. Additionally, three genes with high transcript levels in nymph stage turned out to play a key role in B. tabaci nymph molting by a nanomaterial-promoted RNAi method. Our data could clarify the structures, phylogeny, categories, expression models, and biological functions of B. tabaci chitinase-like family genes, and provide further understanding on insect chitinases, which may advance novel insect pest management strategies.